KR20200008620A - Assay for Plasma Associated Diseases - Google Patents

Abstract

The present application provides a method of identifying or monitoring a plasma cell associated disease, the method comprising purifying an immunoglobulin free light chain (FLC) from a sample from a subject with an anti-FLC specific antibody or fragment thereof and in the sample Applying the purified sample to mass spectrometry techniques to identify the presence of one or more peaks corresponding to one or more monoclonal FLCs.

Description

Assay for Plasma Associated Diseases

The present invention relates to a method for identifying or monitoring a plasma cell associated disease by purifying a free light chain (FLC) from a subject and detecting it using mass spectrometry.

Antibody molecules (also known as immunoglobulins) have double symmetry and typically consist of two identical heavy chains and two identical light chains, each comprising a variable domain and a constant domain. The variable domains of the heavy and light chains combine to form an antigen-binding site, allowing the two chains to contribute to the antigen-binding specificity of the antibody molecule. The basic tetrameric structure of an antibody includes two heavy chains covalently linked by disulfide bonds. Each heavy chain is eventually attached to the light chain again via disulfide bonds. This produces substantially "Y" shaped molecules.

The heavy chain is the larger of the two types of chains found in antibodies, which has a typical molecular weight of 50,000-77,000 Da compared to smaller light chains having a typical molecular weight of 22,000-25,000 Da. Have

There are five main classes or classes or classes of heavy chains, IgG, IgA, IgM, IgD and IgE, which are G, A, M, D and E, which make up the heavy chain. IgG is the major immunoglobulin of normal human serum, which accounts for 70-75% of the total immunoglobulin pool. This is the main antibody of the secondary immune response. It forms a single tetramer of two heavy chains and two light chains.

IgM accounts for approximately 10% of the immunoglobulin pools. Together with the J-chain, the molecule forms five pentamers of basic four-chain structure. The individual heavy chains have a molecular weight of approximately 65,000 Da, and the entire molecule has a molecular weight of about 970,000 Da. IgM is widely confined by vascular pools and is the predominant early antibody.

IgA represents 15-20% of human serum immunoglobulin pools. More than 80% of IgA exists as monomer. However, some of the IgA (secretory IgA) are in dimeric form.

IgD accounts for less than 1% of total plasma immunoglobulins. IgD is found on the surface membrane of mature B-cells.

IgE is rare in normal serum, but is found on the surface membranes of basophils and mast cells. It is associated with allergic diseases such as asthma and hay-fever.

In addition to the five main classes or classes, there are four subclasses for IgG (IgGl, IgG2, IgG3 and IgG4). In addition, there are two subclasses of IgA (IgAl and IgA2).

There are two types of light chains: lambda (λ) and kappa (κ). There are approximately twice as many κ as λ molecules produced in humans, but they are quite different in some mammals. Each chain comprises approximately 220 amino acids in a single polypeptide chain that is folded into one constant domain and one variable domain. Plasma cells produce one of five heavy chain types with either a κ molecule or a λ molecule. Generally about 40% more glass light chains are produced than heavy chain synthesis. If the light chain molecule does not bind to the heavy chain molecule, it is known as a "free light chain molecule". κ light chains are usually found as monomers. λ light chains tend to form dimers.

There are a number of proliferative diseases associated with antibody producing cells.

In many of these proliferative diseases, plasma cells proliferate to form monoclonal tumors of the same plasma cells. It produces large amounts of the same immunoglobulin and is known as monoclonal gamma globulinopathy.

Diseases such as myeloma and primary systemic amyloidosis (AL amyloidosis) account for about 1.5% and 0.3% of cancer mortality rates in the UK, respectively. Multiple myeloma is the second most common form of hematologic malignancy following non-Hodgkin's lymphoma. The incidence in the white population is approximately 40 per million people per year. Typically, the diagnosis of multiple myeloma involves the presence of large amounts of monoclonal plasma cells in the bone marrow, the presence of monoclonal immunoglobulins in serum or urine and associated organ or tissue damage, such as hypercalcemia, kidney failure, anemia or bone lesions. Based on existence. The normal plasma cell content of the bone marrow is about 1% of nucleated cells, whereas in multiple myeloma its content is typically greater than 10%, frequently greater than 30%, but may exceed 90%.

AL amyloidosis is a protein structural disorder characterized by the accumulation of monoclonal free light chain fragments as amyloid deposits. Typically, such patients suffer from heart failure or kidney failure, but peripheral nerves and other organs may also be involved.

There are a number of diseases that can be identified by the presence of monoclonal immunoglobulins in the bloodstream or in fact urine of the patient. This includes plasmacytoma, extramyeloid plasmacytoma, plasmacytoma that occurs outside the bone marrow and can be generated in any organ. If present, the monoclonal protein is typically IgA. Multiple isolated plasmacytomas can occur with or without evidence of multiple myeloma. Waldenstrom's macroglobulinaemia is a low grade lymphoproliferative disorder associated with the production of monoclonal IgM. There are approximately 1,500 new cases annually in the United States and 300 new cases in the United Kingdom. Serum IgM quantitation is important for both diagnosis and monitoring. B-cell non-Hodgkin's lymphoma causes approximately 2.6% of all cancer deaths in the UK and monoclonal immunoglobulins have been identified in the serum of about 10-15% of patients using standard electrophoresis. Initial reports indicate that monoclonal free light chains can be detected in the urine of 60-70% of patients. B-cell chronic lymphocytic leukemia monoclonal protein was identified by free light chain immunoassay.

In addition, there is a so-called MGUS condition. This is the undetermined monoclonal gammopathy of undetermined significance. The term refers to the unexpected presence of monoclonal intact immunoglobulins in individuals without evidence of multiple myeloma, AL amyloidosis, Waldenstrom's macroglobulinemia, and the like. MGUS can be found in 1% of population over 50 years old, 3% over 70 years old, and up to 10% in population over 80 years old. Most of these are related to IgG or IgM, but more rarely IgA-related or bi-clonal. Most people with MGUS die from unrelated diseases, but MGUS can be transformed into malignant monoclonal gamma globulinemia.

In at least some cases for the diseases highlighted above, such diseases exhibit abnormal concentrations of monoclonal immunoglobulins or free light chains. If the disease produces abnormal replication of plasma cells, this often results in the production of more immunoglobulins by the type of cell as the "monoclonal" proliferates and appears in the blood.

Immunofixation electrophoresis uses precipitated antibodies against immunoglobulin molecules. This improves the sensitivity of the test but cannot be used to quantify monoclonal immunoglobulins due to the presence of precipitated antibodies. Immunoelectrophoresis may also be more difficult to perform and difficult to interpret. Capillary zone electrophoresis is used in many clinical laboratories for serum protein separation, which can detect most monoclonal immunoglobulins. However, when compared to immunofixation, capillary region electrophoresis fails to detect monoclonal proteins in 5% of the samples. This so-called "false negative" result includes low concentrations of monoclonal protein.

Total κ and λ assays were generated. However, the total κ and total λ assays are too insensitive to detect monoclonal immunoglobulins or free light chains. This is due to the high background concentration of the polyclonal bound light chain, which interferes with this assay.

More recently, sensitive assays have been developed that can separately detect free κ light chains and free λ light chains. This method uses polyclonal antibodies directed against the free κ light chain or the free λ light chain. The possibility of generating such antibodies has also been discussed in WO 97/17372 as one of a number of different possible specificities. This document discloses a method of tolerising an animal to allow the production of the desired antibody more specific than the prior art can produce. Free light chain assays use antibodies to bind to free λ or free κ light chains. The concentration of the free light chain is determined by nephelometry or turbidimetry. This involves adding the test sample to a solution containing the appropriate antibody in a reaction vessel or cuvette. As the beam of light passes through the cuvette and the antigen-antibody reaction proceeds, the light passing through the cuvette is scattered more and more because an insoluble immune complex is formed. In the turbidity method, light scattering is monitored by measuring the light intensity at an angle away from the angle of incidence, while in the turbidity measurement light scattering is monitored by measuring the decrease in the intensity of the incident beam of light. A series of calibrator of known antigen (ie free κ or free I) concentration is first assayed to generate a calibration curve of light scatter measurements versus antigen concentration.

This assay form was found to successfully detect free light chain concentrations. In addition, the sensitivity of these techniques is very high.

Characterization of the amount or type of free-light chain (FLC), heavy chain or subclass, or light chain-type bound to heavy chain class or subclass has been characterized by B cell diseases such as multiple myeloma and other immune mediated diseases such as nephropathy. It is important in a wide range of diseases, including nephropathy.

WO2015 / 154052 (Mayo Foundation), which is hereby incorporated by reference in its entirety, discloses methods for detecting immunoglobulin light chains, immunoglobulin heavy chains, or mixtures thereof using mass spectrometry (MS). Samples comprising immunoglobulin light chains, heavy chains or mixtures thereof are immunopurified and reduced to separate the light and heavy chains and subjected to mass spectrometry to obtain mass spectra of the samples. It can be used to detect monoclonal proteins in samples from patients. It can also be used to fingerprint, isotype and identify post-translational modifications such as disulfide bonds and glycosylation in monoclonal antibodies.

WO 2015/131169 (H. Lee Moffitt Cancer Center) describes a method for monitoring conditions associated with abnormal antibody production. It uses enzymatic cleavage of the target immunoglobulin and measures one or more variable domain peptide fragments by quantitative mass spectrometry. This method is complex because it relies on the identification of variable domain peptide fragments specific to the specific target immunoglobulin associated with the disease and is associated with long enzymatic cleavage.

Applicants of the present invention have recognized that free light chain (FLC) specific antibodies and mass spectrometry (MS) can be used in selective and rapid assays to identify plasma cell associated diseases. Unexpectedly, we found that the technique is sensitive enough to detect FLC in normal patients (95% normal standard range for free kappa light chain is 3.3-19.4 mg / L, 95% for free lambda light chain) Normal standard range is 5.7-26.3 mg / L). The prior art considers significantly higher levels of immunoglobulins such as IgG (adult normal levels are typically 6-16 g / L).

Anti-FLC antibodies are used to purify FLC from the sample to reduce contaminants in the assay. Applicants have recognized that when purified FLCs from normal samples are analyzed by MS, they produce curves of different sizes and differently charged FLCs. 1 shows typical results for anti-free lambda, FIG. 2 shows typical results for anti-free kappa, and FIGS. 3 and 4 show overlap and summed curves of size for normal kappa and lambda production. This curve consists of a number of different individual peaks of FLC produced by normal antibody producing cells in healthy subjects. Applicant has recognized that in monoclonal disease there is an increased amount of FLC (s) due to amplification of the clone (s). This increases the amount of FLC significantly higher than background normal FLC production. This can be perceived as a sharp peak in intensity (see FIG. 5). It also increases the sensitivity of the assay compared to prior art systems.

Typically, the ratio of kappa and lambda is measured to identify abnormal FLC production. This requires separate measurements and accurate quantification of kappa and lambda FLCs to obtain their ratios. This also depends on the amount of one of kappa and lambda being increased relative to the other type of chain, the other being distorted, for example when there are multiple clones.

The present invention does not require separating kappa FLC and lambda FLC to be quantified. Instead it depends on single detection of increased peaks compared to background FLC production. This allows, for example, high sensitivity to achieve identification of nonsecretory multiple myeloma and AL amyloidosis. It is also possible to reduce intact immunoglobulins to allow FLC to be determined without releasing the light chain bound to the heavy chain.

FLC is present at significantly lower concentrations, such as less than 40 mg / L, such as about 26 mg / L, compared to intact immunoglobulins (typically 6-16 g / L) in adults. The 95% normal reference range for the free kappa light chain is 3.3 to 19.4 mg / L and for free lambda light chain is 5.7 to 26.3 mg / L. It is therefore surprising that FLC can be identified even in normal patients and the presence of monoclonal FLC in MGUS patients.

The present invention is directed to separating an immunoglobulin free light chain (FLC) from a sample from a subject having an anti-FLC specific antibody or fragment thereof and to identifying the presence of one or more peaks corresponding to one or more monoclonal FLCs in the sample. A method of identifying or monitoring a plasma cell associated disease is provided that includes subjecting the purified sample to mass spectrometry techniques.

That is, mass spectrometry assays typically separate glass light chains due to charge and mass when applied to mass spectrometry. This typically produces a normal distribution of FLCs with different molecular weights that reflect the molecular weight of germline light chain amino acid sequences and somatic hypermutations of such sequences in subjects with normal FLCs. As discussed above, the presence of monoclonal FLCs produces peaks resulting from increased amounts of monoclonal FLCs produced by plasma cell associated diseases. Monoclonal FLCs are sized and charged and are identified by an increased amount (peak) relative to the background normal FLC present.

Mass spectrometry (MS), as used herein, is, for example, liquid chromatography-mass spectrometry (LC-MS), microfluidic liquid chromatography electrospray ionization (coupled to quadrupole flight time mass spectrometry). liquid chromatography electrospray ionisation coupled to a quadruple time-of-flight mass spectrometry (micro LC-ESI-Q-TOF MS). This may include, for example, the use of the positive ion mode. Orbitlab mass spectrometers, ion trap mass spectrometers, flight time mass spectrometers, triple quadrupole mass spectrometers, or quadrupole mass spectrometers can be used.

Alternatively, MS technology includes matrix assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF-MS). When MALDI-TOF is used, it is typically used in positive mode for charged ions, preferably 1+, 2+ and / or 3+ ions, most preferably 2+ ions.

Typically enzymatic cleavage of the FLC is not performed.

The anti-FLC specific antibody or fragment thereof may be a monoclonal or polyclonal antibody or fragment. The antibody may be a synthetic antibody, and synthetic antibodies include recombinant antibodies, nucleic acid aptamers and non-immunoglobulin protein scaffolds.

The antibody may be species specific, such as anti-human or anti-horse or anti-sheep or anti-pig. Antibodies can be generated in cartilage fish, sheep, goats, horses, rabbits, cattle, llamas, rats or mice. The antibody or fragment can specifically bind to the free light chain.

The fragment of the antibody can be, for example, an F (ab ') ₂ fragment.

Anti-FLC specific antibodies or fragments may be anti-kappa FLC specific or anti-lambda FLC specific. That is, lambda FLC and kappa FLC can be separated separately from the sample. For example, two separate assays will then be performed by mass spectrometry. Thereafter, separate lambda FLC records and separate kappa FLC records, such as those shown in FIGS. 1 and 2, will be produced.

Alternatively, or more typically, anti-kappa FLC specific antibodies or fragments and anti-lambda specific antibodies or fragments are used. This co-purifies the lambda glass light chain and the kappa glass light chain to produce the readings shown in FIG. 4, for example for normal healthy patients. Monoclonal peaks are still identified above the combined background FLC.

An anti-FLC specific antibody or fragment thereof may comprise one or more non-disulfide bridges between at least one heavy chain (or fragment) and at least one light chain (or fragment) of the antibody or fragment thereof.

Crosslinking typically includes thioether bonds. Alternative crosslinks may also be used.

Thioether bridges include thioether bonds. This is the linkage between the residues of the antibody, where this linkage has a single sulfur bond rather than a disulfate bond. That is, thioether bridges do not include linkages containing more than one sulfur atom, such as disulfide bridges familiar to those skilled in the art. Instead, thioether bridges include one sulfur bond that bridges the residues of the macromolecule. One or more additional non-sulfur atoms may further form a link.

The residues linked by thioether bridges can be natural or non-natural residues. Formation of thioether bridges can result in the loss of atoms from residues as will be appreciated by those skilled in the art. For example, the formation of a thioether bridge between the side chains of two cysteine residues can result in the loss of sulfur and hydrogen atoms from the residue, but those skilled in the art will recognize that the resulting thioether bridges link cysteine residues.

Thioether crosslinking may connect any two residues of an antibody. One or more of the residues may be selected from, for example, cysteine, aspartic acid, glutamic acid, histidine, methionine and tyrosine. Two of the residues may be selected from the group consisting of cysteine, aspartic acid, glutamic acid, histidine, methionine and tyrosine. More typically two of the residues are cysteine residues. Typically, only one thioether bridge is present between the heavy and light chains. Alternatively, two, three or more thioether crosslinks can be used. Heavy chain pairs of an antibody, or fragment thereof, may also be linked by one or more non-disulfide bridges, such as thioether bonds.

Thioether crosslinks are described, for example, in WO2006 / 099481, and Zhang et al (2013) J. Biol. Chem. vol 288 (23), 16371-8 and Zhang & Flynn (2013) J. Biol. Chem, vol 288 (43), 34325-35.

Phosphine and phosphite can be used. As used herein, 'phosphine' refers to any compound comprising at least one functional unit having the general formula R ₃ P, wherein P is phosphorus and R is any other atom. In phosphites, the R position is specifically occupied by oxygen atoms. R ₃ P-containing compounds act as strong nucleophiles capable of attacking disulfide bonds. This may result in the reduction of sulfides, but under some conditions may also result in thioether bond formation.

Compounds include:

Tris (dimethylamino) phosphine (CAS No. 1608-26-0)

Tris (diethylamino) phosphine (CAS No. 2283-11-6)

Trimethylphosphite (CAS No. 121-45-9)

Tributylphosphine (CAS No. 998-40-3)

References: Bernardes et al. (2008) Angew. Chem. Int. Ed. , vol 47, 2244-2247, incorporated herein by reference.

Crosslinking may also include crosslinking agents, such as maleimide crosslinking agents, which react with the free thiol to crosslink the chain of the antibody molecule. It may be bound on one side of the thiol group, further on another moiety such as lysine carboxyl group, as described in WO00 / 44788.

Bifunctional crosslinkers comprising two reactive moieties linked together by a linker, in particular a flexible linker, can be used. Such linkers may comprise one or more carbons covalently bonded together in a chain, eg, substituted or unsubstituted alkyl. Such linkers are in particular C1-C10, most typically C2-C6 or C3-C6 linkers. Applicants believe that due to the relatively high recovery levels of C2-C6 containing crosslinkers such as α, α'-dibromo- m -xylene, BMOE (bismaleimidoethane) or BMB (bismaleimidobutane) It was found to be particularly useful.

Bismaleimide is a homobifunctional sulfhydryl reactive crosslinker.

This is a widely characterized class of crosslinkers comprising two maleimide groups linked by hydrocarbons or other crosslinkers. Maleimide groups covalently crosslink the two remaining cystine by spontaneously reacting with the free sulfhydryl groups exposed by reduction of disulfide to form a non-reducing thioether bond in each sulfhydryl.

Compounds include:

Bis (maleimido) ethane (CAS number 5132-30-9)

l, 4-bis (maleimido) butane (CAS No. 28537-70-4)

references:

Auclair et al . (2010) Strategies for stabilizing superoxide dismutase (SOD1), the protein destabilized in the most common form of familial amyotrophic lateral sclerosis. Proc Natl Acad Sci USA , vol 107 (50)-pages 21394-9

Geula et al . (2012) Structure-based analysis of VDAC1 protein: defining oligomer contact sites. J Biol Chem , vol 287 (3), 475-85

Kida et al . (2007) Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis. J Cell Biol , vol 171 (7) pages 1441-1452, incorporated herein by reference.

α, α′-dibromo-m-xylene is a homobifunctional sulfhydryl reactive crosslinker that may also be used.

Dibromo- m -xylene (CAS No. 626-15-3) is one of the di-alkyl halide systems and acts as a homofunctional crosslinker that reacts with free sulfhydryl groups.

References :

Jo et al . (2012) Development of α-Helical Calpain Probes by Mimicking a Natural Protein-Protein Interaction J Am Chem Soc. , col 134 (42)-pages 17704-13, incorporated herein by reference.

Alternative sulfhydryl reactive crosslinking compounds to form stable thioether bonds

There are at least six classes of reagents known to react with free sulfhydryls resulting in non-reducing covalent crosslinked products. The specific reactivity of these compounds to sulfhydryl groups varies and some will react with water, amine and carboxyl groups under certain conditions. In addition, many of these compounds have bulky linker groups, which can limit the ability to crosslink in limited space environments. The following list provides some examples from each class, but a more comprehensive list and references are provided below:

Chemistry of Protein Conjugation and Cross-linking, Wong, S: ISBN 0-8493-5886-8, incorporated herein by reference.

Bismaleimide

Bis (maleimido) hexane; N-N'-methylenebismaleimide; Bis (N-maleimidomethyl) ether; N, N '-(1,3-phenylene) -bismaleimide; Bis (N-maleimido) -4,4'-bibenzyl; Naphthalene-1,5-dimaleimide

Haloacetyl derivatives

1,3-dibromoacetone; N, N'-bis (iodoacetyl) polymethylenediamine; Ν, Ν'- di (bromoacetyl) phenylhydrazine; 1,2-di (bromoacetyl) amino-3-phenylhydrazine; γ- (2,4-dinitrophenyl) -α-bromoacetyl-L-diaminobutyrate bromoacetyl-hydrazide

Di-alkyl halides

α, α'-dibromo- p -xylene sulfonic acid; α, α'-diiodo- p -xylene sulfonic acid; Di (2-chloroethyl) sulfide; Tri (2-chloroethyl) amine; N, N-bis (β-bromoethyl) benzylamine

2.4 s Triazine

Dichloro-6-methoxy- s -triazine; 2,4,6-trichloro- s -triazine (cyanuric acid); 2,4-dichloro-6- (3'-methyl-4-aminoanilino) -s -triazine; 2,4-dichloro-6-amino- s -triazine

Aziridine

2,4,6-tri (ethyleneimido) -s -triazine; N, N'-ethyleneiminoyl-1,6-diaminohexane; Tri [1- (2-methylaziridenyl)]-phosphine oxide

Bis-epoxide

1,2: 3,4-diepoxybutane; 1,2: 5,6-diepoxyhexane; Bis (2, -epoxypropyl) ether; 1,4-butadiol diglycidoxyether

Crosslinking may replace one or more naturally occurring disulfide bonds, or alternatively may be produced in addition to disulfide bonds.

Other crosslinking chemicals may also be included. Alternative crosslinking may include:

Carboxyl vs. Primary Amine

(a) Carbodiimide Activation: N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide HCI (EDC; CAS Nr 25952-53-8.

(b) Carbodiimide activation: EDC stabilized with N-hydroxysulfosuccinimide (sNHS; CAS Nr 106627-54-7)

(c) 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium (DMTMM; CAS Nr 3945-69-5)

ㆍ When activated with any of the above, the carboxylate will react with the primary amine (lysine, N-terminus) to form a covalent amide bond.

ㆍ Note: Under some conditions, EDC / sNHS activation can lead to the formation of covalent ester bonds between activated carboxyl groups (ie, aspartic acid, glutamic acid, C-terminus) and hydroxyl groups (ie, serine, threonine and tyrosine).

Carboxyl vs carboxyl

(a) carboxyl is activated with EDC, EDC / sNHS or DMTMM, and then crosslinked with amine-derivatized polyethylene glycol, eg amine-PEGn-amine, where n is the number of repeat PEG units

(b) carboxyl activation with DMTMM and crosslinking with allofunctional hydrazides such as adipic dihydrazide (ADH; CAS Nr 1071-93-8)

Carboxyl vs sulfhydryl

If disulfide is present, a reducing agent, for example. Reduced to -SH with tris (2-carboxyethyl) phosphine hydrochloride (TCEP; CAS Nr 51805-45-9). Carboxyl is activated with carbodiimide (EDC) and crosslinked with:

(a) 3- (4- (4- (aminomethyl) -1H-1,2,3-triazol-1-yl) phenyl) propiononitrile hydrochloride (APN; CAS Nr 1643841-88-6)

(b) amine- (PEG) n-maleimide, where n is the number of repeating PEG units (MAL-PEG-NH2)

Amines versus amines

(a) PEGylated bis (sulfosuccinimidyl) suvrate), for example BS (PEG) 5, where 5 is the number of repeat PEG units

(b) Dimethyl pimelimate (DMP; CAS Nr 58537-94-3)

(c) p -phenylene diisothiocyanate (PDITC; CAS Nr 4044-65-9)

(d) Subic acid bis (N-hydroxysuccinimide ester) (DSS; CAS Nr 68528-80-3)

(e) ethylene glycol bis (sulfosuccinimidyl succinate) (sulfo-EGS; CAS Nr 167410-92-6)

Amines vs sulfhydryl

(a) Maleimide-PEG8-succinimidyl ester (CAS Nr 756525-93-6)

(b) 4- (N-maleimidomethyl) cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC; CAS Nr 64987-85-5)

(c) 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP; CAS Nr 68181-17-9)

(d) 3- (4-formylphenyl) propiononitrile (APN-CHO)

(e) Iodoacetic acid N-hydroxysuccinimide ester (IAA-NHS; CAS Nr 39028-27-8)

Hydroxyl vs sulfhydryl

(a) 4- (maleimido) phenyl isocyanate (PMPI; CAS Nr 123457-83-0)

Other chemicals

(a) p -azidophenylglyoxal (APG; CAS Nr 1196151-49-1)

ㆍ Reacts with arginine and, to a lesser extent, cystine (disulfide bond) and histidine. Upon photoactivation, the addition reaction is initiated using a primary amine or through a double bond, C-H and N-H via a ring expansion mechanism.

(b) 1,4-butanediol diglycidyl ether (BDDE; CAS Nr 2425-79-8)

ㆍ Reacts with hydroxyl, amine and sulfhydryl groups

(c) 4- (4-diazoniophenyl) benzenediazonium (CAS Nr 5957-03-9)

ㆍ Reacts with tyrosine and histidine

(d) Benzophenone-4-iodoacetamide (CAS Nr 76809-63-7)

ㆍ Reacts with sulfhydryl and forms covalent bonds upon photoactivation with active C-H and N-H bonds

(e) succinimidyl 2-[(4,4'-azippentaneamido) ethyl] -1,3'-dithiopropionate) (SDAD; CAS Nr 1253202-38-8)

ㆍ NHS ester groups react with primary amines; Diazirin groups upon photoactivation react efficiently with any amino acid side chain or peptide backbone.

Typically at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the antibodies are crosslinked. For example, using bismaleimide, crosslinking efficiencies of 70% to 80% were observed. For example, the addition of a reducing agent can disrupt the disulfide bonds of the remaining non-crosslinked antibodies and isolate them using, for example, gel electrophoresis, thereby further purifying the crosslinked antibodies to produce higher levels of crosslinking. Can be.

An advantage of using a crosslinked antibody is that it reduces the contamination of the sample by, for example, the free light chain released from the purified antibody. This increases the sensitivity and accuracy of the system.

The method comprises purifying whole kappa and whole lambda light chains with anti-whole kappa antibodies or fragments thereof and anti-whole lambda antibodies or fragments thereof, and identifying the presence of one or more peaks corresponding to monoclonal light chain production. Applying the sample to the mass spectrometry may further comprise. That is, the anti-FLC specific antibody binds substantially only the free light chain, while the whole kappa lambda light chain and / or the whole lambda light chain also binds both the free light chain and the light chain that binds the heavy chain. It therefore detects not only the free light chain, but any light chain present in the sample. This additional step, for example, helps to identify MGUS from which monoclonal intact immunoglobulins are produced.

Again, separate whole kappa light chain-specific antibodies and whole lambda light chain-specific antibodies can be used, or alternatively a mixture of the two antibodies can be used together. Antibodies and fragments can be as defined above or modified as above. Typically this is crosslinked with one or more non-disulfide crosslinks.

An alternative method that can be used instead of or in addition to assaying with whole kappa and / or lambda antibodies is to apply a portion of the sample to reducing conditions using, for example, a reducing agent. This releases the bound light chain from the heavy chain. The released light chain can then be concentrated or purified using anti-whole kappa and / or lambda light chains or using anti-kappa and / or anti-lambda FLC antibodies.

If the antibody used in the concentration step is modified by the presence of one or more non-sulfide crosslinks between the light and heavy chains of the detection antibody, it is still used under reducing conditions to be released in the sample as shown in FIGS. 9 and 10. Light chains can be detected.

The sample can be a suitable body fluid, including, for example, tears, plasma, serum, blood, urine, saliva or cerebrospinal fluid.

Plasma cell associated diseases can be any where the disease produces one or more monoclonal light chains. These include, for example: intact immunoglobulins, multiple myeloma, light chain multiple myeloma, nonsecretory multiple myeloma, AL amyloidosis, light chain deposition disease (LCDD), asymptomatic multiple myeloma, undetermined monoclonal gamma globulinemia (MGUS) , Macroglobulinemia, POEMS (peripheral neuropathy, organ hypertrophy, endocrine disease, monoclonal gamma globulin disease and skin changes) syndrome.

Antibodies or fragments used in the present invention to purify FLC or whole light chains may be provided on suitable immunopurification columns of, for example, the type generally known in the art. Alternatively, the antibody can be attached to magnetic beads of the type known for example as "DynaBeads ™". This allows the antibody to mix with the sample and bind to the FLC or the entire light chain in the sample. Antibodies attached to the FLC or light chain may then be removed from the sample with the aid of a magnet to which the antibody or fragment is attracted.

The FLC or light chain can then be eluted from the antibody and used in the mass spectrometer, for example by placing it on a mass spectrometry target.

Alternatively, the antibody or fragment can be immobilized on, for example, a mass spectrometry target. The sample is contacted with a target comprising an antibody, the target is washed to remove unbound material, and then a mass spectrometry target comprising bound FLC or light chain (via the fragment of the antibody) is subjected to mass spectrometry to bind The presence of free light chain or light chain is detected.

Accordingly, further aspects of the invention provide anti-kappa FLC antibodies or fragments thereof, anti-lambda FLC antibodies or fragments thereof, and at least one mass spectrometry target. Typically antibodies are immobilized on mass spectrometry targets. Typically a mixture of anti-kappa FLC and anti-lambda FLC antibodies is immobilized on the target.

Also provided is a mass spectrometer comprising a mass spectrometry target as defined above.

The methods of the invention can also be used to further characterize any condition a subject has in combination with one or more additional assays. For example:

Serum plasma electrophoresis or immunofixation electrophoresis may be performed to further characterize the condition.

Total protein albumin or beta-2-microglobulin can be detected by MS or conventional assays known in the art.

Renal function markers such as creatinine and cystatin can be assayed. Heart markers such as troponin, NT-pro-BNP can also be assayed. Bone profile / turnover for hypercalcemia can be assayed as alkaline phosphatase (ALP) and phosphate (Ph).

Accordingly, the claimed invention detects a variety of different plasma cell associated diseases, including intact immunoglobulins, multiple myeloma, light chain multiple myeloma, nonsecretory multiple myeloma, AL amyloidosis, light chain deposition, asymptomatic multiple myeloma, plasmacytoma and MGUS. It is predicted. MGUS will be detected if a normal FLC clone is present.

The table below shows the sensitivity of the most relevant symptom-related monoclonal gamma globulin disease screening panel compared to the predicted sensitivity of the present invention. SPE is serum plasma electrophoresis, sFLC is serum free light chain, and MS FLC is the present invention.

The invention will now be described by way of example only, with reference to the following figures:
1 shows mass spectrometry performed after purification with anti-free lambda antibodies in positive ion mode covering a single charged ion range of 22.5 to 23.5 kDa of a normal sample without plasma cell associated disease.
2 shows mass spectrometry conducted in positive ion mode covering a single charged ion range of 22.5 to 23.5 kDa of normal samples after purification with anti-free kappa antibody.
3 shows the overlap of the outputs for FIGS. 1 and 2.
4 shows the effect of co-purification with anti-free lambda and anti-free kappa antibodies on normal samples.
5 shows mass spectrometry performed with abnormal samples after purification with anti-free lambda. This shows abnormal peaks indicating the presence of abnormal monoclonal proteins.
FIG. 6 shows mass spectrometry execution of abnormal samples in the presence of abnormal clonal production of free lambda after purification of anti-free kapparo.
FIG. 7 shows the superposition of the outputs shown in FIGS. 5 and 6.
FIG. 8 shows mass spectrometry execution of abnormal samples comprising abnormal clonal production of free lambda after co-purification of anti-free lambda and anti-free kapparo.
9 shows crosslinking of both anti-human IgG antibodies by BS (PEG) ₅ , as shown by the reduced SDS-PAGE assay. L ₁ = free immunoglobulin light chain, H ₁ = free immunoglobulin heavy chain, H ₁ L ₁ and H ₂ L ₂ = crosslinked heavy and light chain moieties.
10 shows that antibodies crosslinked with BS (PEG) ₅ retain biological activity. Both anti-human IgG antibodies were crosslinked using increasing concentrations of BS (PEG) ₅ and analyzed for their IgG binding activity by ELISA.
11A shows mass spectrometry performed on samples from subjects with IgG kappa MGUS in positive ion mode for double charged ions.
FIG. 11B shows mass spectrometry performed on samples from subjects with IgG kappa MGUS in positive ion mode for single charged ions.
12A shows mass spectrometry performed on samples from subjects with IgA lambda MGUS in positive ion mode for double charged ions.
12B shows mass spectrometry performed on samples from subjects with IgA lambda MGUS in positive ion mode for single charged ions.
FIG. 13A shows mass spectrometry performed on samples from subjects with IgA lambda MGUS in positive mode for double charged ions.
13B shows mass spectrometry performed on samples from subjects with IgA lambda MGUS in positive ion mode for single charged ions.
14A shows mass spectrometry performed from normal subjects in a positive ion mode for double charged ions.
FIG. 14B shows a mass spectrometry implementation of the normal sample of FIG. 14A in positive ion mode for single charged ions.
15A shows a mass spectrometry run of normal samples in positive ion mode for double charged ions.
FIG. 15B shows mass spectrometry implementation of the normal sample of FIG. 15A in positive ion mode for single charged ions.

Kappa FLC and Lambda FLC may be purified separately or co-purified using anti-kappa FLC antibodies and anti-lambda FLC antibodies, or mixtures thereof.

Purified FLC is spotted on mass spectrometry plates and analyzed by MALDI-TOF.

1-8 show how the presence of a monoclonal free light chain in the serum of a patient can be readily identified by the presence of a peak on normal production of the background of the free light chain. Such peaks can be identified even when there is overlap between the kappa peak and the lambda peak.

Anti-human IgG antibodies can be crosslinked by the allofunctional crosslinker BS (PEG) ₅ .

Antibodies were incubated with increasing concentrations of BS (PEG) ₅ (0-40 molar excess) and assayed by reducing SDS-PAGE assay. As shown in FIG. 9, in the absence of BS (PEG) ₅ , a reducing agent (50 mM DTT) causes the antibody to dissociate into its heavy and light chain portions. In contrast, incubation of the antibody with increasing concentrations (from 0 to 40 molar excess) of BS (PEG) ₅ increases the crosslinking of the heavy and light chains to form a reduced-tolerant heavy chain-light chain pair.

Antibodies crosslinked with BS (PEG) ₅ retain biological activity.

Purified human IgG lambda was coated at 3 to 2000 ng / mL on microtiter plates. After crosslinking with 0-40 molar excess of BS (PEG) ₅ , both anti-human IgG antibodies were applied. The amount of bound antibody was determined using a horse radish peroxidase reporter enzyme and a donkey anti-sheep antibody conjugated to a 3,3 ', 5,5'-tetramethylbenzidine chromogenic substrate. As shown in FIG. 10, no significant effects on human IgG binding were observed at concentrations of BS (PEG) ₅ up to 40 × molar excess when compared to uncrosslinked antibodies.

Testing of Samples and Normal Samples from Subjects with MGUS

Human serum samples (three IPE positive MGUS Figures 11-13 (a-c) and two healthy controls Figures 14 and 15) were placed in PBS-T buffer (25 mM sodium phosphate, 150 mM NaCl, 0.1% Tween). (tween 20), pH 7.0), incubated with antibody coated magnetic beads, resuspended, and then washed sequentially with 3 x and deionized water in PBS-T. Beads were eluted with acid buffer for 15 minutes at RT. One of the eluates was mixed with the matrix (α-cyano-4-hydroxycinnamic acid, 10 mg / gl), then spotted using a Mosquito HTS spotter (TTP) on a polished steel MALDI target plate and Analyze on Bruker Biotyper MALDI-TOF mass spectrometer (Microflex LT / SH Smart). Positive ion mode covering a range of 10,000 to 30,000 m / z including single charged (+1, m / z 22 to 25 kDa) ions and double charged (= 2, m / z 10 to 14 kDa) ions Mass spectra were obtained. Data was analyzed using Bruker Flex analysis software.

This indicates that mass spectrometry can be used to detect monoclonal kappa glass light and lambda glass light chains even at the level of free light chains generally recognized in normal samples.

Preliminary results also indicate that an abnormal monoclonal FLC can be detected better than a single positive mode using a positive mode for double charged ions.

In samples, even in subjects with MGUS that are often difficult to identify, the ability to quickly identify the presence of low levels of FLC is surprising and subjects with MGUS and subjects with other monoclonal gamma globulin It opened the way for new approaches that could be identified.

Claims (14)

A method of identifying or monitoring plasma cell associated diseases, comprising: purifying an immunoglobulin free light chain (FLC) from an sample from a subject with an anti-FLC specific antibody or fragment thereof and at least one monoclonal in the sample Applying the purified sample to mass spectrometry techniques to identify the presence of one or more peaks corresponding to FLC. The method of claim 1, wherein the anti-FLC specific antibody is a mixture of an anti-kappa FLC specific antibody or fragment thereof and an anti-lambda FLC specific antibody or fragment thereof. The method of claim 1 or 2, wherein the anti-FLC specific antibody or fragment is polyclonal. The method of any one of claims 1-3, wherein the antibody fragment is an F (ab ') ₂ fragment. The method of claim 1, wherein the anti-FLC specific antibody or fragment is one between at least one heavy chain (or fragment) and one light chain (or fragment) of the antibody or fragment thereof. The non-disulfide cross-link comprising the above. 6. The mass spectrometry technique of claim 1, wherein the mass spectrometry techniques that can be used include orbitrap mass spectrometers, ion trap mass spectrometers, time-of-flight mass spectrometers. Or, triple quadrupole mass spectrometer, or quadrupole mass spectrometer. The method of claim 6, wherein the mass spectrometry technique is matrix assisted laser desorption ionisation-time-of-flight (MALDI-TOF). 8. The method according to claim 1, wherein the whole kappa light chain and the whole lambda light chain are purified with anti-whole kappa antibodies or fragments and whole anti-lambda antibodies or fragments and correspond to at least one monoclonal immunoglobulin. Applying the purified sample to mass spectrometry to identify one or more peaks. The method of claim 8, wherein the whole lambda light chain and the whole kappa light chain are co-purified using a mixture of anti-whole kappa antibody and anti-whole lambda antibody. The method of claim 1, wherein the plasma cell associated disease is intact immunoglobulin, multiple myeloma, light chain multiple myeloma, nonsecretory multiple myeloma, AL amyloidosis, light chain deposition disease (LCDD). , Asymptomatic multiple myeloma, undetermined monoclonal gammopathy of undetermined significance (MGUS), macroglobulinemia, POEMS (peripheral neuropathy, long-term hypertrophy, endocrine disease, monoclonal gamma globulin) ) Syndrome and LCDD. The method of claim 1, wherein the sample is tear, plasma, serum, saliva, urine, blood or cerebrospinal fluid. Anti-kappa free light chain antibodies, fragments thereof; Anti-lambda free light chain antibodies thereof; And a mass spectrometry target. The kit of claim 12, wherein the antibody is immobilized on the mass spectrometry target. A mass spectrometer comprising a mass spectrometry target according to claim 13.

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